RGMa collapses the neuronal actin barrier against disease-implicated protein and exacerbates ALS.

Repulsive guidance molecule A (RGMa) was originally identified as a neuronal growth cone-collapsing factor. Previous reports have demonstrated the multifunctional roles of RGMa mediated by neogenin1. However, the pathogenic involvement of RGMa in amyotrophic lateral sclerosis (ALS) remains unclear. Here, we demonstrated that RGMa concentration was elevated in the cerebrospinal fluid of both patients with ALS and transgenic mice overexpressing the mutant human superoxide dismutase1 (mSOD1 mice). Treatment with humanized anti-RGMa monoclonal antibody ameliorated the clinical symptoms in mSOD1 mice. Histochemical analysis revealed that the anti-RGMa antibody significantly decreased mutant SOD1 protein accumulation in the motor neurons of mSOD1 mice via inhibition of actin depolymerization. In vitro analysis revealed that the anti-RGMa antibody inhibited the cellular uptake of the mutant SOD1 protein, presumably by reinforcing the neuronal actin barrier. Collectively, these data suggest that RGMa leads to the collapse of the neuronal actin barrier and promotes aberrant protein deposition, resulting in exacerbation of the ALS pathology.

Adult-onset botulism in a Japanese woman with prolonged spore excretion.

We report a case of an 80-year-old woman with botulism from 2020 in Osaka, Japan. The patient complained of dysarthria and dizziness. On the same day, the patient developed respiratory failure, and was intubated and placed on mechanical ventilation. Subsequently, ophthalmoparesis and quadriparesis progressed rapidly. Ten days after onset, the patient failed to respond to any external stimulation. Blood tests showed anemia, and computed tomography revealed undiagnosed cervical cancer. Initially, diagnosis of neuromuscular junction disorder and acute motor neuropathy, including paraneoplastic syndrome, were considered. However, intravenous immunoglobulin therapy and plasma exchange were ineffective. A fecal sample on day 30 showed a large number of C. botulinum spores. On day 34, a mouse bioassay revealed botulinum toxin type A in the patient's serum; therefore, a botulinum antitoxin was administered. Later, the patient's muscle strength was gradually improved. However, severe muscle paralysis persisted, and the patient died of cachexia owing to cervical cancer on day 196. The etiology of this case was unknown because no contaminated food was identified during an inspection of the patient's home. Fecal 16S rRNA gene sequencing revealed dysbiosis of the intestinal microbiota with abundant Enterococcus species. Long-lasting excretion of substantial botulinum spores even on day 30 indicated colonization of C. botulinum in the intestinal tract. This case suggests that C. botulinum colonization with co-existing intestinal dysbiosis may be associated with severe and prolonged symptoms of botulism.

Single-cell RNA-seq analysis identifies distinct myeloid cells in a case with encephalitis temporally associated with COVID-19 vaccination.

Recently accumulating evidence has highlighted the rare occurrence of COVID-19 vaccination-induced inflammation in the central nervous system. However, the precise information on immune dysregulation related to the COVID-19 vaccination-associated autoimmunity remains elusive. Here we report a case of encephalitis temporally associated with COVID-19 vaccination, where single-cell RNA sequencing (scRNA-seq) analysis was applied to elucidate the distinct immune signature in the peripheral immune system. Peripheral blood mononuclear cells (PBMCs) were analyzed using scRNA-seq to clarify the cellular components of the patients in the acute and remission phases of the disease. The data obtained were compared to those acquired from a healthy cohort. The scRNA-seq analysis identified a distinct myeloid cell population in PBMCs during the acute phase of encephalitis. This specific myeloid population was detected neither in the remission phase of the disease nor in the healthy cohort. Our findings illustrate induction of a unique myeloid subset in encephalitis temporally associated with COVID-19 vaccination. Further research into the dysregulated immune signature of COVID-19 vaccination-associated autoimmunity including the cerebrospinal fluid (CSF) cells of central nervous system (CNS) is warranted to clarify the pathogenic role of the myeloid subset observed in our study.

Cell-Free DNA Derived From Neutrophils Triggers Type 1 Interferon Signature in Neuromyelitis Optica Spectrum Disorder.

BACKGROUND AND OBJECTIVES: Recently accumulating evidence suggests the pivotal role of type 1 interferon (IFN-1) signature in the pathogenesis of neuromyelitis optica spectrum disorder (NMOSD). However, the mechanism of the initial trigger that augments IFN-1 pathway in the peripheral immune system of NMOSD has yet to be elucidated. METHODS: Clinical samples were obtained from 32 patients with aquaporin-4 antibody-positive NMOSD and 23 healthy subjects. IFN-1 induction in peripheral blood mononuclear cells (PBMCs) by serum-derived cell-free DNA (cfDNA) was assessed in combination with blockades of DNA sensors in vitro. CfDNA fraction was analyzed for DNA methylation profiles by bisulfite sequencing, elucidating the cellular origin of cfDNA. The induction of neutrophil extracellular trap related cell death (NETosis) was further analyzed in NMOSD and control groups, and the efficacy of pharmacologic intervention of NETosis was assessed. RESULTS: Enhanced IFN-1 induction by cfDNA derived from NMOSD was observed in PBMCs with cofactor of LL37 antimicrobial peptide. DNase treatment, cGAS inhibitor, and Toll-like receptor 9 antagonist efficiently inhibited IFN-1 production. DNA methylation pattern of cfDNA in patients with NMOSD demonstrated that the predominant cellular source of cfDNA was neutrophils. Whole blood transcriptome analysis also revealed neutrophil activation in NMOSD. In addition, enhanced NETosis induction was observed with NMOSD-derived sera, and efficient pharmacologic inhibition of NETosis with dipyridamole was observed. DISCUSSION: Our study highlights the previously unrevealed role of cfDNA predominantly released by neutrophil in the induction of IFN-1 signature in NMOSD and further indicate a novel pharmacologic target in NMOSD.

High cell surface expression and peptide binding affinity of HLA-DQA1*05:03, a susceptible allele of neuromyelitis optica spectrum disorders (NMOSD).

Neuromyelitis optica spectrum disorder (NMOSD) is a relapsing autoimmune disease characterized by the presence of pathogenic autoantibodies, anti-aquaporin 4 (AQP4) antibodies. Recently, HLA-DQA1*05:03 was shown to be significantly associated with NMOSD in a Japanese patient cohort. However, the specific mechanism by which HLA-DQA1*05:03 is associated with the development of NMOSD has yet to be elucidated. In the current study, we revealed that HLA-DQA1*05:03 exhibited significantly higher cell surface expression levels compared to other various DQA1 alleles, and that its expression strongly depended on the amino acid sequence of the α1 domain, with a preference for leucine at position 75. Moreover, in silico analysis indicated that the HLA-DQ encoded by HLA-DQA1*05:03 preferentially presents immunodominant AQP4 peptides, and that the peptide major histocompatibility complexes (pMHCs) are more energetically stable in the presence of HLA-DQA1*05:03 than other HLA-DQA1 alleles. In silico 3D structural models were also applied to investigate the validity of the energetic stability of pMHCs. Taken together, our findings indicate that HLA-DQA1*05:03 possesses a distinct property to play a pathogenic role in the development of NMOSD.